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Yeast two-hybrid (Y2H) technology is one of the most widely applied methods for detecting protein–protein interactions (PPIs) in vivo, and it has played a central role in molecular biology, signaling pathway analysis, disease mechanism research, and drug target discovery. With advantages such as operational simplicity, scalable screening capacity, and compatibility with intracellular environments, Y2H has evolved from the classical nuclear system into multiple specialized platforms, including membrane-associated systems, cytosolic interaction systems, split-protein sensors, and high-throughput sequencing–coupled screening models. These expansions enable researchers to investigate increasingly complex protein classes and construct comprehensive interaction networks.
The nuclear yeast two-hybrid (Y2H) system represents the earliest and most widely established form of Y2H technology. Its logic is built on the modular structure of eukaryotic transcriptional activators. In the canonical system, the transcription factor GAL4 is divided into two distinct functional domains:
(1) the DNA-binding domain (BD), which recognizes upstream activating sequences (UAS);
(2) the activation domain (AD), which drives transcriptional initiation of downstream reporter genes.
In Y2H, the BD and AD are separately fused to two proteins of interest-the bait and prey. When the bait and prey interact, the BD and AD are brought into close spatial proximity, reconstituting a functional GAL4 transcription factor that activates reporter gene expression. Common reporter systems include HIS3, ADE2, and LacZ, enabling detection through auxotrophic selection or enzymatic colorimetric assays.
Figure 1. Schematic representation of GAL4-mediated transcriptional activation.
In practical screening workflows, the bait fusion (BD–X) is co-expressed with prey fusion proteins (AD–Y), either individually or as part of a cDNA library. If an interaction occurs, reporter genes are activated; if not, the reporter remains silent. For the identification of unknown interacting partners, cDNA libraries fused to the AD are introduced into yeast cells, enabling high-throughput discovery of novel prey proteins.
Figure 2. Classical nuclear Y2H principle diagram.
The nuclear Y2H system features clear readouts and is highly suitable for large-scale screening. However, it has intrinsic limitations, particularly for proteins with intrinsic transcriptional activity, proteins localized outside the nucleus, or membrane proteins requiring specific subcellular environments. These challenges have driven the development of expanded Y2H formats.
Traditional Y2H reactions occur in the nucleus, rendering it unsuitable for many membrane proteins or proteins whose biological functions depend on membrane-associated topology. To address these limitations, researchers developed the split-ubiquitin system, forming the foundation of the membrane-based yeast two-hybrid (MbYTH) platform.
In this system, ubiquitin (Ub) is divided into two parts:
• Nub (N-terminal half)
• Cub (C-terminal half), which is fused to a transcription factor (TF) to form Cub–TF
The commonly used NubG variant contains a point mutation (Ile13→Gly or Ala) to reduce spontaneous reassociation with Cub. When bait (X) fused to Cub–TF interacts with prey (Y) fused to NubG within the membrane environment, NubG and Cub reassemble into a quasi-native ubiquitin structure. This reconstructed ubiquitin is recognized by ubiquitin-specific proteases (UBPs), which cleave and release TF. The liberated TF then translocates into the nucleus and activates reporter gene transcription.
Figure 3. Schematic diagram of membrane protein Y2H.
MbYTH is particularly valuable for studying full-length membrane proteins, multi-pass transporters, receptors, and membrane-associated signaling complexes. It enables detection within the native membrane milieu, overcoming the limitations of nuclear localization required by classical Y2H.
However, additional experimental complexity arises, notably the requirement to consider membrane topology. Depending on whether the N- or C-terminus of a protein faces the cytoplasm or extracellular space, the orientation of NubG or Cub–TF may not allow productive reassociation. Thus, cDNA libraries must be constructed in both orientations (NubG–X and X–NubG) to enable comprehensive screening, particularly for type I and type II membrane proteins.
As research targets broadened to include transcription factors, cytosolic complexes, and structurally sensitive proteins, several improved Y2H variants were developed, including cytosolic Y2H (cytoY2H) and split-protein reconstitution systems, such as the split-tryptophan (Trp) sensor.
1. Cytosolic Y2H (cytoY2H): Restricting Bait Localization to the ER Membrane
The cytoY2H system is based on the same split-ubiquitin principle as MbYTH but introduces the membrane-anchoring protein Ost4p to immobilize the bait on the endoplasmic reticulum (ER) membrane. This prevents transcriptionally active proteins from entering the nucleus and artificially activating reporters. Prey proteins fused to NubG remain cytosolic, and only genuine interactions enable Cub–NubG reassociation, releasing the LexA–VP16 transcription factor for nuclear translocation and reporter activation.
cytoY2H expands applicability to many protein classes that are difficult or impossible to analyze via classical Y2H, including transcription factors and proteins with strong activation domains.
Figure 4. Schematic of the cytoY2H system. (Möckli et al., 2007)
2. Split-Tryptophan Sensor System: A Direct Enzyme-Reconstitution Method
Inspired by the split-ubiquitin concept, researchers also created alternative split-protein reporters. One notable example is the split-tryptophan (Trp1p) system, in which functional N-terminal (NTrp) and C-terminal (CTrp) fragments of Trp1p only reassemble when their fused partner proteins interact. Reconstitution of Trp1p enzymatic activity enables yeast growth on tryptophan-deficient media.
This system offers several advantages:
• direct detection independent of transcriptional pathways;
• no requirement for nuclear localization;
• compatibility with diverse protein types;
• orientation-independent reconstitution.
Figure 5. Split-tryptophan fragment identification and fusion strategy. (Tafelmeyer P et al., 2024)
Traditional Y2H workflows rely on manual colony isolation and Sanger sequencing, which become labor-intensive and costly in large-scale interactome studies. With the advent of high-throughput sequencing (HTS), researchers developed Y2H-seq and CrY2H-seq, enabling exhaustive interaction mapping at an unprecedented scale.
1. Y2H-seq: Bulk Sequencing of All Positive Interactions
Y2H-seq combines classical Y2H screening with next-generation sequencing by pooling all positive colonies from selective media and extracting their plasmids as a mixture. Sequencing the pooled DNA identifies all prey sequences that interact with a given bait, while read abundance provides semi-quantitative information on interaction strength.
Figure 6. Workflow diagram of the Y2H-seq process. (Erffelinck et al., 2018)
2. CrY2H-seq: Cre/LoxP-Mediated Genomic Recording of Interaction Pairs
To enable library–library screening, Cre-recombinase–assisted Y2H sequencing (CrY2H-seq) employs bait and prey plasmids flanked by lox66 and lox71 sites. Upon Cre transcription induced by interaction, recombination covalently fuses the bait and prey ORFs into a single contiguous DNA fragment. These fused products represent direct records of interacting protein pairs and are amplified via PCR and sequenced. This method greatly reduces cost and increases throughput in interactome mapping.
Figure 7. CrY2H-seq plasmid design and screening workflow. (Möckli et al., 2007)
Successful Y2H experiments rely on meticulous execution of multiple steps-yeast competent-cell preparation, transformation, selection, interaction verification, RNA extraction, mRNA purification, and cDNA library construction. Key procedures include:
1. Preparation of Yeast Competent Cells
Competent cells must be prepared at optimal physiological states (typically OD600 ≈ 0.6) to ensure high transformation efficiency. Parameters such as temperature, shaking speed, centrifugation force, and resuspension quality directly impact downstream success.
2. Yeast Transformation and Interaction Verification
The PEG/LiAc transformation method remains the standard for introducing plasmids into yeast. Heat shock, DMSO treatment, and incubation timing must be carefully controlled. Interaction verification is performed using selective media and/or colorimetric responses, with stringent controls to avoid false positives or bait autoactivation.
3. Construction of High-Complexity Yeast cDNA Libraries
Library construction typically involves:
• total RNA extraction (e.g., CTAB method),
• mRNA purification via magnetic beads,
• cDNA synthesis,
• adaptor ligation,
• size fractionation,
• Gateway or SMART-based recombination,
•E. coli DH10B transformation,
• library capacity estimation and insert-size validation.
Reliable library complexity and representation are crucial for successful downstream interaction screening.
High-quality yeast cDNA libraries enable researchers to perform PPI screening against bait proteins, reconstruct interaction networks, and infer biological functions of unknown genes. Their applications include:
• discovery of novel interacting partners;
• mapping of protein–protein interaction networks;
• elucidation of signaling pathways;
• identification of disease-associated proteins in cancer, neurodegeneration, and rare disorders;
• guiding drug target discovery and validation.
Coupled with high-throughput sequencing, Y2H-based discovery is progressively transforming from pairwise detection to systems-level interactome profiling.
We provide end-to-end Y2H services, from bait and prey construct design to large-scale library screening and data interpretation. Our portfolio includes classical nuclear Y2H, membrane and cytosolic variants, split-protein systems, and sequencing-enabled Y2H workflows tailored to diverse protein classes and research objectives.
Built for Challenging Targets
Our optimized yeast strains, flexible vector architectures, and curated library options allow reliable analysis of transcription factors, membrane-associated proteins, signaling components, and transient or low-affinity interactions that are difficult to capture using conventional assays.
High-Throughput, Data-Rich Discovery
By integrating next-generation sequencing with Y2H screening, Creative Biogene enables quantitative, high-throughput interaction mapping across complex libraries. Sequencing-driven analytics improve sensitivity, reduce background noise, and support scalable interactome studies.
From Interaction Maps to Insight
We go beyond interaction detection. Creative Biogene supports downstream validation, network analysis, and functional annotation, helping transform Y2H results into biologically meaningful pathways, mechanisms, and actionable research leads.
Designed for Discovery and Translation
Whether for target identification, pathway analysis, or mechanism-of-action studies, our Y2H services are designed to integrate seamlessly into broader discovery pipelines, supporting both fundamental research and translational programs.
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